1. Field of the Invention
This invention relates to methods of detecting G-protein-coupled receptor (GPCR) activity, and provides methods of assaying GPCR activity and methods for screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process.
The actions of many extracellular signals are mediated by the interaction of G-protein-coupled receptors (GPCRs) and guanine nucleotide-binding regulatory proteins (G-proteins). G-protein-mediated signaling systems have been identified in many divergent organisms, such as mammals and yeast. The GPCRs represent a large super family of proteins which have divergent amino acid sequences, but share common structural features, in particular, the presence of seven transmembrane helical domains. GPCRs respond to, among other extracellular signals, neurotransmitters, hormones, odorants and light. Individual GPCR types activate a particular signal transduction pathway; at least ten different signal transduction pathways are known to be activated via GPCRs. For example, the beta 2-adrenergic receptor (β2AR) is a prototype mammalian GPCR. In response to agonist binding, β2AR receptors activate a G-protein (Gs) which in turn stimulates adenylate cyclase activity and results in increased cyclic adenosine monophosphate (cAMP) production in the cell.
The signaling pathway and final cellular response that result from GPCR stimulation depends on the specific class of G-protein with which the particular receptor is coupled (Hamm, “The many faces of G-Protein Signaling.” J. Biol. Chem., 273:669-672 (1998)). For instance, coupling to the Gs class of G-proteins stimulates cAMP production and activation of Protein Kinase A and C pathways, whereas coupling to the Gi class of G-proteins down regulates cAMP. Other second messenger systems as calcium, phospholipase C, and phosphatidylinositol 3 may also be utilized. As a consequence, GPCR signaling events have predominantly been measured via quantification of these second messenger products.
A common feature of GPCR physiology is desensitization and recycling of the receptor through the processes of receptor phosphorylation, endocytosis and dephosphorylation (Ferguson, et al., “G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins.” Can. J. Physiol. Pharmacol., 74:1095-1110 (1996)). Ligand-occupied GPCRs can be phosphorylated by two families of serine/threonine kinases, the G-protein-coupled receptor kinases (GRKs) and the second messenger-dependent protein kinases such as protein kinase A and protein kinase C. Phosphorylation by either class of kinases serves to down-regulate the receptor by uncoupling it from its corresponding G-protein. GRK-phosphorylation also serves to down-regulate the receptor by recruitment of a class of proteins known as the airestins that bind the cytoplasmic domain of the receptor and promote clustering of the receptor into endocytic vescicles. Once the receptor is endocytosed, it will either be degraded in lysosomes or dephosphorylated and recycled back to the plasma membrane as fully-functional receptor.
Binding of an arrestin protein to an activated receptor has been documented as a common phenomenon for a variety of GPCRs ranging from rhodopsin to β2AR to the neurotensin receptor (Barak, et al., “A β-arrestin/Green Fluorescent fusion protein biosensor for detecting G-Protein-Coupled Receptor Activation,” J. Biol. Chem., 272:27497-500 (1997)). Consequently, monitoring arrestin interaction with a specific GPCR can be utilized as a generic tool for measuring GPCR activation. Similarly, a single G-protein and GRK also partner with a variety of receptors (Hamm, et al. (1998) and Pitcher et al., “G-Protein-Coupled Receptor Kinases,” Annu. Rev. Biochem., 67:653-92 (1998)), such that these protein/protein interactions may also be monitored to determine receptor activity.
The present invention involves the use of a proprietary technology (ICAST™, Intercistronic Complementation Analysis Screening Technology) for monitoring protein/protein interactions in GPCR signaling. The method involves using two inactive β-galactosidase mutants, each of which is fused with one of two interacting protein pairs, such as a GPCR and an arrestin. The formation of an active β-galactosidase complex is driven by interaction of the target proteins. In this system, β-galactosidase activity acts as a read out of GPCR activity. FIG. 23 is a schematic depicting the method of the present invention. FIG. 23 shows two inactive mutants that become active when they interact. In addition, this technology could be used to monitor GPCR-mediated signaling pathways via other downstream signaling components such as G-proteins, GRKs or c-Src.
Many therapeutic drugs in use today target GPCRs, as they regulate vital physiological responses, including vasodilation, heart rate, bronchodilation, endocrine secretion and gut peristalsis. See, e.g., Lefkowitz et al., Annu. Rev. Biochem., 52:159 (1983). For instance, drugs targeting the highly studied GPCR, β2AR are used in the treatment of anaphylaxis, shock hypertension, asthma and other conditions. Some of these drugs mimic the ligand for this receptor. Other drugs act to antagonize the receptor in cases when disease arises from spontaneous activity of the receptor.
Efforts such as the Human Genome Project are identifying new GPCRs (“orphan” receptors) whose physiological roles and ligands are unknown. It is estimated that several thousand GPCRs exist in the human genome.